How to formulate hypotheses and IATAs to support grouping and read-across of nanoforms.

Manufacturing and functionalizing materials at the nanoscale has led to the generation of a whole array of nanoforms (NFs) of substances varying in size, morphology, and surface characteristics. Due to financial, time, and ethical considerations, testing every unique NF for adverse effects is virtually impossible. Use of hypothesis-driven grouping and read-across approaches, as supported by the GRACIOUS Framework, represents a promising alternative to case-by-case testing that will make the risk assessment process more efficient. Through application of appropriate grouping hypotheses, the Framework facilitates the assessment of similarity between NFs, thereby supporting grouping and read-across of information, minimizing the need for new testing, and aligning with the 3R principles of replacement, reduction, and refinement of animals in toxicology studies. For each grouping hypothesis an integrated approach to testing and assessment (IATA) guides the user in data gathering and acquisition to test the hypothesis, following a structured format to facilitate efficient decision-making. Here we present the template used to generate the GRACIOUS grouping hypotheses encompassing information relevant to "Lifecycle, environmental release, and human exposure", "What they are: physicochemical characteristics", "Where they go: environmental fate, uptake, and toxicokinetics", and "What they do: human and environmental toxicity". A summary of the template-derived hypotheses focusing on human health is provided, along with an overview of the IATAs generated by the GRACIOUS project. We discuss the application and flexibility of the template, providing the opportunity to expand the application of grouping and read-across in a logical, evidence-based manner to a wider range of NFs and substances.

An Integrated Testing Strategy for Ecotoxicity (ITS-ECO) Assessment in the Marine Environmental Compartment using Mytilus spp.: A Case Study using Pristine and Coated CuO and TiO2 Nanomaterials.

An integrated testing strategy for ecotoxicity assessment (ITS-ECO) was developed to aid in the hazard and fate assessment of engineered nanomaterials (ENMs) deposited in marine environments using the bivalve Mytilus spp. as a test species. The ENMs copper(II) oxide (CuO) and titanium dioxide (TiO2 ), either in pristine form (core) or with functionalized coatings (polyethylene glycol [PEG], carboxyl [COOH], and ammonia [NH3 ]) were selected as case study materials based on their production levels and use. High-throughput in vitro testing in Tier 1 of the ITS-ECO revealed CuO ENMs to elicit cytotoxic effects on lysosomes of hemocytes of mussels, with the hazard potential CuO PEG > CuO COOH > CuO NH3 > CuO core, whereas TiO2 ENMs were not cytotoxic. Genotoxicity in hemocytes as well as gill cells of mussels following in vivo exposure (48 h) to CuO ENMs was also seen. Longer in vivo exposures in Tier 2 (48 h-21 days) revealed subacute and chronic oxidative effects for both CuO and TiO2 ENMs, in some cases leading to lipid peroxidation (core TiO2 ENMs). In Tier 3 bioaccumulation studies, distinct patterns of uptake for Cu (predominantly in gills) and Ti (predominantly in digestive glands) and between the different core and coated ENMs were found. Clear NM-specific and coating-dependent effects on hazard and fate were seen. Overall, using a tiered testing approach, the ITS-ECO was able to differentiate the hazard (acute, subacute, and chronic effects) posed by ENMs of different compositions and coatings and to provide information on fate for environmental risk assessment of these ENMs. Environ Toxicol Chem 2022;41:1390-1406. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Acute waterborne and chronic sediment toxicity of silver and titanium dioxide nanomaterials towards the oligochaete, Lumbriculus variegatus.

The use of silver (Ag) and titanium dioxide (TiO2) nanomaterials (NMs) in industrial processes and consumer products has experienced considerable growth since the late 20th century. Throughout their lifecycle, both Ag NM and TiO2NM are released into the environment, with benthic systems anticipated to be the final sink. Their potential toxicity towards benthic species is therefore of major concern. This study investigated the toxicity of silver (Ag; NM-300 K) and titanium dioxide (TiO2; NM-104) NMs to the freshwater oligochaete, Lumbriculus variegatus in acute (0-96-h) waterborne and chronic (28-d) sediment studies. Toxicity was investigated via assessment of mortality, behaviour, and antioxidant enzyme activity. The 96-h LC50 for Ag NMs in water was 0.51 mg/l (95% CI, 0.45-0.56), with L. variegatus displaying inhibited predation-avoidance behaviour compared to controls (6.66 ± 10%) successful response at 24-h), as well as significant increases (p < 0.05) in catalase (CAT) activity at sub-lethal concentrations at 24-h. Behavioural improvement and the return of antioxidant enzymes to control levels was observed after 48 and 72-h. AgNO3 exposure proved more toxic than Ag NM (96-h LC50 = 0.034 mg/l, 95% CI, 0.031-0.037) but resulted in no changes to antioxidant enzymes following sub-lethal exposure. Furthermore, Ag dissolution from Ag NM (~2-4%) could not account for the full extent of toxicity observed, suggesting a nano-specific effect. Increased environmental relevance via the inclusion of Suwannee River Humic Acid (SRHA, 5 mg/l) alleviated sub-lethal Ag NM toxicity despite a comparable 96-h LC50 (0.54 mg/l, 95% CI, 0.51-0.57). Significant effects of Ag NMs in formulated sediments (mortality, biomass) were only recorded according to OECD 225 at the highest test concentration (1333 mg/kg) for Ag NM indicating a potential attenuating effect of sediments towards toxicity. No toxicity was observed for TiO2 NM in aquatic or sediment exposures up to concentrations of 2000 mg/l and 1333 mg/kg, respectively.

Risk Management Framework for Nano-Biomaterials Used in Medical Devices and Advanced Therapy Medicinal Products.

The convergence of nanotechnology and biotechnology has led to substantial advancements in nano-biomaterials (NBMs) used in medical devices (MD) and advanced therapy medicinal products (ATMP). However, there are concerns that applications of NBMs for medical diagnostics, therapeutics and regenerative medicine could also pose health and/or environmental risks since the current understanding of their safety is incomplete. A scientific strategy is therefore needed to assess all risks emerging along the life cycles of these products. To address this need, an overarching risk management framework (RMF) for NBMs used in MD and ATMP is presented in this paper, as a result of a collaborative effort of a team of experts within the EU Project BIORIMA and with relevant inputs from external stakeholders. The framework, in line with current regulatory requirements, is designed according to state-of-the-art approaches to risk assessment and management of both nanomaterials and biomaterials. The collection/generation of data for NBMs safety assessment is based on innovative integrated approaches to testing and assessment (IATA). The framework can support stakeholders (e.g., manufacturers, regulators, consultants) in systematically assessing not only patient safety but also occupational (including healthcare workers) and environmental risks along the life cycle of MD and ATMP. The outputs of the framework enable the user to identify suitable safe(r)-by-design alternatives and/or risk management measures and to compare the risks of NBMs to their (clinical) benefits, based on efficacy, quality and cost criteria, in order to inform robust risk management decision-making.

Exposure to Pb-halide perovskite nanoparticles can deliver bioavailable Pb but does not alter endogenous gut microbiota in zebrafish.

Lead-halide perovskite nanoparticles (NPs) are a new technology, and investigation of toxicity is of considerable importance due to the potential lead (Pb) release into the environment. The aim of the study was to investigate aqueous and dietary toxicity of Pb-halide perovskite NP and Pb in zebrafish Danio rerio. Perovskite NP toxicity was evaluated in zebrafish by mortality, gene expression, histopathology, and phylogenetic analysis of gut microbiota. Zebrafish larvae were exposed to five Pb-halide perovskite NPs in parallel with Pb(NO3)2 exposures, and zebrafish adults were exposed to the three perovskite NPs that caused the strongest effect and Pb(NO3)2. No median lethal concentration (LC50) was observed for zebrafish larvae exposed to up to 200 mg/L of perovskite NPs for 96 h. Mortality, metallothionein 2 (mt2) and δ-aminolevulinic acid dehydratase (ala-d) gene expression (24-h exposure) in zebrafish larvae after aqueous perovskite NPs exposures did not differ from total Pb concentration - response curves. The lack of differences in mortality and gene expression between perovskite NPs and soluble Pb after aqueous exposure suggest that toxicity from perovskite NPs can be attributed to bioavailable Pb rather than nano-specific effects. Induction of mt2 and reduction of ala-d expression levels in liver tissues showed Pb bioavailability after 2-d and 4-d dietary exposure to perovskite-spiked feeds. Changes in gut microbiota of adult zebrafish were detected after 14-d exposure to Pb-spiked food, but no changes were detected from perovskite-NP spiked food. The phylogenetic analysis identified different microbiome profiles of Pb-fed fish compared to perovskite-fed fish suggesting a different mechanism of toxicity. Exposure to Pb-halide perovskite NPs led to absorption of Pb likely from release of Pb ions rather than absorption of NPs. Pb-halide perovskite NPs can release bioavailable Pb and this needs to be considered during the development of this technology.

Importance of Surface Coating to Accumulation Dynamics and Acute Toxicity of Copper Nanomaterials and Dissolved Copper in Daphnia magna.

We evaluated the effect of copper oxide nanomaterials (CuO NMs), uncoated and with 3 different surface coatings (carboxylated, pegylated, and ammonia groups), on acute toxicity and accumulation dynamics in Daphnia magna. With the use of biodynamic modelling, biosorption and elimination rate constants were determined for D. magna following waterborne exposure to dissolved Cu and CuO NMs. The relationship between modeled parameters and acute toxicity endpoints was evaluated to investigate whether accumulation dynamics parameters could be used as a predictor of acute toxicity. The Langmuir equation was used to characterize the biosorption dynamics of Cu NMs and Cu chloride, used as dissolved Cu control. Uptake rates showed the following NM rankings: pristine-CuO > NH3 -CuO > aqueous Cu > polyethylene glycol (PEG)-CuO > COOH-CuO. To determine Cu elimination by D. magna, a one-compartment model was used. Different elimination rate constants were estimated for each chemical substance tested. Those that were easily biosorbed were also easily removed from organisms. Biosorption and depuration properties of NMs were correlated with zeta potential values and diameters of NM agglomerates in the suspensions. No link was found between biosorption and toxicity. Waterborne exposures to more difficult-to-biosorb CuO NMs were more likely to induce adverse effects than those that biosorbed easily. It is proposed that some physicochemical properties of NMs in media, including zeta potential and agglomerate diameter, can lead to higher biosorption but do not necessarily affect toxicity. The mode of interaction of the NMs with the organism seems to be complex and to depend on chemical speciation and physicochemical properties of the NMs inside an organism. Moreover, our findings highlight that coating type affects the biosorption dynamics, depuration kinetics, and dissolution rate of NMs in media. Environ Toxicol Chem 2020;39:287-299. © 2019 SETAC.

The influence of organic modification on the cytotoxicity of clay particles to keratinocytes, hepatocytes and macrophages; an investigation towards the safe use of polymer-clay nanocomposite packaging.

Organically modified clays can be used as nanofillers in polymer-clay nanocomposites to create bio-based packaging with improved strength and barrier properties. The impact of organic modification on the physico-chemical properties and toxicity of clays has yet to be fully investigated but is essential to ensure their safe use. Two organoclays, named N116_HDTA and N116_TMSA, were prepared using a commercially available sodium bentonite clay and the organic modifiers hexadecyl trimethyl ammonium bromide (HDTA) and octadecyl trimethyl ammonium chloride (TMSA). An in vitro hazard assessment was performed using HaCaT skin cells, C3A liver cells, and J774.1 macrophage-like cells. Organic modification with HDTA and TMSA increased the hazard potential of the organoclays in all cell models, as evidenced by the higher levels of cytotoxicity measured. N116_TMSA caused the greatest loss in viability with IC50 values of 3.2, 3.6 and 6.1 µg/cm2 calculated using J774.1, HaCaT and C3A cell lines, respectively. Cytotoxic effects were dictated by the amount of free or displaced organic modifier present in the exposure suspensions. The parent bentonite clay also caused distinct cytotoxic effects in J774.1 macrophage-like cells with associated TNF-α release. Such information on the hazard profile of organoclays, can feed into risk assessments for these materials.

Differences in Engineered Nanoparticle Surface Physicochemistry Revealed by Investigation of Changes in Copper Bioavailability During Sorption to Nanoparticles in the Aqueous Phase.

Sorption of chemical substances to nanoparticles (NPs) in the aqueous phase strongly influences NP physicochemisty, and investigations of these complex interactions can provide important insights into the environmental fate of NPs. The objective of the present study was to use differences in copper (Cu) bioavailability to investigate aqueous-phase sorption with NPs that had different physicochemical characteristics (silicon [Si], perovskite, and titanium dioxide NPs [TiO2 NPs]). Sorption of Cu with NPs was assessed by the presence of adsorbent in water and onto the NP surface after ultracentrifugation, and by changes in Cu bioavailability under static conditions during exposure of larval zebrafish, as well as under conditions of continuous agitation during exposure of the alga Chlorella vulgaris. The presence of TiO2 NPs reduced total Cu in the water column and Cu bioavailability (measured by growth inhibition, mortality, and metallothionein 2 gene expression), confirming Cu sorption to TiO2 NPs. Nanoparticle surface area was the most important factor that affected Cu sorption, as indicated by less bioavailable Cu in the presence of smaller TiO2 NPs. The surface area effect was consistent regardless of exposure conditions (alga, continuous agitation; zebrafish, static water) and was further supported by the fact that the lowest total Cu concentration in the water column was found in the presence of the smallest NP. The results differed with other NP types, for example, silicon NPs, in which Cu sorption was indicated by analytical chemistry, but sorption was not sufficient to significantly alter Cu bioavailability. The bioavailability tests did not indicate Cu sorption with perovskite NPs. The results demonstrate that surface area critically influences sorption, that Cu sorption as measured by bioavailability is not affected by agitation or static conditions, and that Cu sorption differs among types of NPs, indicating differences in their surface physicochemistry. Environ Toxicol Chem 2019;9999:1-11. © 2019 SETAC.

Toward sustainable environmental quality: Priority research questions for Europe.

The United Nations' Sustainable Development Goals have been established to end poverty, protect the planet, and ensure prosperity for all. Delivery of the Sustainable Development Goals will require a healthy and productive environment. An understanding of the impacts of chemicals which can negatively impact environmental health is therefore essential to the delivery of the Sustainable Development Goals. However, current research on and regulation of chemicals in the environment tend to take a simplistic view and do not account for the complexity of the real world, which inhibits the way we manage chemicals. There is therefore an urgent need for a step change in the way we study and communicate the impacts and control of chemicals in the natural environment. To do this requires the major research questions to be identified so that resources are focused on questions that really matter. We present the findings of a horizon-scanning exercise to identify research priorities of the European environmental science community around chemicals in the environment. Using the key questions approach, we identified 22 questions of priority. These questions covered overarching questions about which chemicals we should be most concerned about and where, impacts of global megatrends, protection goals, and sustainability of chemicals; the development and parameterization of assessment and management frameworks; and mechanisms to maximize the impact of the research. The research questions identified provide a first-step in the path forward for the research, regulatory, and business communities to better assess and manage chemicals in the natural environment. Environ Toxicol Chem 2018;37:2281-2295. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

The Essential Elements of a Risk Governance Framework for Current and Future Nanotechnologies.

Societies worldwide are investing considerable resources into the safe development and use of nanomaterials. Although each of these protective efforts is crucial for governing the risks of nanomaterials, they are insufficient in isolation. What is missing is a more integrative governance approach that goes beyond legislation. Development of this approach must be evidence based and involve key stakeholders to ensure acceptance by end users. The challenge is to develop a framework that coordinates the variety of actors involved in nanotechnology and civil society to facilitate consideration of the complex issues that occur in this rapidly evolving research and development area. Here, we propose three sets of essential elements required to generate an effective risk governance framework for nanomaterials. (1) Advanced tools to facilitate risk-based decision making, including an assessment of the needs of users regarding risk assessment, mitigation, and transfer. (2) An integrated model of predicted human behavior and decision making concerning nanomaterial risks. (3) Legal and other (nano-specific and general) regulatory requirements to ensure compliance and to stimulate proactive approaches to safety. The implementation of such an approach should facilitate and motivate good practice for the various stakeholders to allow the safe and sustainable future development of nanotechnology.

Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials.

Assessing the safety of engineered nanomaterials (NMs) is paramount to the responsible and sustainable development of nanotechnology, which provides huge societal benefits. Currently, there is no evidence that engineered NMs cause detrimental health effects in humans. However, investigation of NM toxicity using in vivo, in vitro, in chemico, and in silico models has demonstrated that some NMs stimulate oxidative stress and inflammation, which may lead to adverse health effects. Accordingly, investigation of these responses currently dominates NM safety assessments. There is a need to reduce reliance on rodent testing in nanotoxicology for ethical, financial and legislative reasons, and due to evidence that rodent models do not always predict the human response. We advocate that in vitro models and zebrafish embryos should have greater prominence in screening for NM safety, to better align nanotoxicology with the 3Rs principles. Zebrafish are accepted for use by regulatory agencies in chemical safety assessments (e.g. developmental biology) and there is growing acceptance of their use in biomedical research, providing strong foundations for their use in nanotoxicology. We suggest that investigation of the response of phagocytic cells (e.g. neutrophils, macrophages) in vitro should also form a key part of NM safety assessments, due to their prominent role in the first line of defense. The development of a tiered testing strategy for NM hazard assessment that promotes the more widespread adoption of non-rodent, alternative models and focuses on investigation of inflammation and oxidative stress could make nanotoxicology testing more ethical, relevant, and cost and time efficient.

Surfactants from itaconic acid: Toxicity to HaCaT keratinocytes in vitro, micellar solubilization, and skin permeation enhancement of hydrocortisone.

One of the most widely used approaches for improving drug permeation across the stratum corneum barrier of the skin is the use of chemical penetration enhancers, such as surfactants. In this study, two anionic surfactants, named C12-OPK and C18-OPK, were synthesized via condensation of itaconic acid and fatty amines, with C12 and C18 alkyl chains, respectively. Assessment of impacts on HaCaT keratinocyte cell viability was used as indicator of their potential to cause skin irritation 24h post exposure (Alamar Blue assay). The LC50 values of C12-OPK and C18-OPK (144 and 85mg/L, respectively) were lower than LC50 values of the most used commercial surfactants (e.g. SDS). The effect of different surfactant concentrations (up to ten times the critical micellar concentration, CMC) on hydrocortisone (HC) solubility and permeation through porcine skin was also evaluated. Results showed that drug solubility increased linearly with increasing concentrations of both surfactants, as a consequence of the association between drug and micelles. In vitro permeation results showed that the permeability coefficient increased at surfactant concentrations lower than the CMC. In particular, a higher enhancement effect on drug permeation was obtained with C18-OPK, due to its hydrophobic properties that ensured a more effective HC permeation in comparison to C12-OPK.

Regulatory ecotoxicity testing of nanomaterials - proposed modifications of OECD test guidelines based on laboratory experience with silver and titanium dioxide nanoparticles.

Regulatory ecotoxicity testing of chemicals is of societal importance and a large effort is undertaken at the OECD to ensure that OECD test guidelines (TGs) for nanomaterials (NMs) are available. Significant progress to support the adaptation of selected TGs to NMs was achieved in the context of the project MARINA ( http://www.marina-fp7.eu/ ) funded within the 7th European Framework Program. Eight OECD TGs were adapted based on the testing of at least one ion-releasing NM (Ag) and two inert NMs (TiO2). With the materials applied, two main variants of NMs (ion releasing vs. inert NMs) were addressed. As the modifications of the test guidelines refer to general test topics (e.g. test duration or measuring principle), we assume that the described approaches and modifications will be suitable for the testing of further NMs with other chemical compositions. Firm proposals for modification of protocols with scientific justification(s) are presented for the following tests: growth inhibition using the green algae Raphidocelis subcapitata (formerly: Pseudokirchneriella subcapitata; TG 201), acute toxicity with the crustacean Daphnia magna (TG 202), development toxicity with the fish Danio rerio (TG 210), reproduction of the sediment-living worm Lumbriculus variegatus (TG 225), activity of soil microflora (TGs 216, 217), and reproduction of the invertebrates (Enchytraeus crypticus, Eisenia fetida, TGs 220, 222). Additionally, test descriptions for two further test systems (root elongation of plants in hydroponic culture; test on fish cells) are presented. Ecotoxicological data obtained with the modified test guidelines for TiO2 NMs and Ag NM and detailed method descriptions are available.

Considerations of Environmentally Relevant Test Conditions for Improved Evaluation of Ecological Hazards of Engineered Nanomaterials.

Engineered nanomaterials (ENMs) are increasingly entering the environment with uncertain consequences including potential ecological effects. Various research communities view differently whether ecotoxicological testing of ENMs should be conducted using environmentally relevant concentrations-where observing outcomes is difficult-versus higher ENM doses, where responses are observable. What exposure conditions are typically used in assessing ENM hazards to populations? What conditions are used to test ecosystem-scale hazards? What is known regarding actual ENMs in the environment, via measurements or modeling simulations? How should exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? These questions were addressed in the context of this critical review. As a result, three main recommendations emerged. First, researchers should improve ecotoxicology of ENMs by choosing test end points, duration, and study conditions-including ENM test concentrations-that align with realistic exposure scenarios. Second, testing should proceed via tiers with iterative feedback that informs experiments at other levels of biological organization. Finally, environmental realism in ENM hazard assessments should involve greater coordination among ENM quantitative analysts, exposure modelers, and ecotoxicologists, across government, industry, and academia.

Pseudomonas putida biofilm dynamics following a single pulse of silver nanoparticles.

Pseudomonas putida mono-species biofilms were exposed to silver nanoparticles (Ag NPs) in artificial wastewater (AW) under hydrodynamic conditions. Specifically, 48 h old biofilms received a single pulse of Ag NPs at 0, 0.01, 0.1, 1, 10 and 100 mg L(-1) for 24 h in confocal laser scanning microscopy (CLSM) compatible flow-cells. The biofilm dynamics (in terms of morphology, viability and activity) were characterised at 48, 72 and 96 h. Consistent patterns were found across flow-cells and experiments at 48 h. Dose dependent impacts of NPs were then shown at 72 h on biofilm morphology (e.g. biomass, surface area and roughness) from 0.01 mg L(-1). The microbial viability was not altered below 10 mg L(-1) Ag NPs. The activity (based on the d-glucose utilisation) was impacted by concentrations of Ag NPs equal and superior to 10 mg L(-1). Partial recovery of morphology, viability and activity were finally observed at 96 h. Comparatively, exposure to Ag salt resulted in ca. one order of magnitude higher toxicity when compared to Ag NPs. Consequently, the use of a continuous culture system and incorporation of a recovery stage extends the value of biofilm assays beyond the standard acute toxicity assessment.

Nanomaterials in the aquatic environment: A European Union-United States perspective on the status of ecotoxicity testing, research priorities, and challenges ahead.

The European Union-United States Communities of Research were established in 2012 to provide a platform for scientists to develop a "shared repertoire of protocols and methods to overcome nanotechnology environmental health and safety (nanoEHS) research gaps and barriers" (www.us-eu.org/). Based on work within the Ecotoxicology Community of Research (2012-2015) the present Focus article provides an overview of the state of the art of nanomaterials (NMs) in the aquatic environment by addressing different research questions, with a focus on ecotoxicological test systems and the challenges faced when assessing NM hazards (e.g., uptake routes, bioaccumulation, toxicity, test protocols, and model organisms). The authors' recommendation is to place particular importance on studying the ecological effects of aged/weathered NMs, as-manufactured NMs, and NMs released from consumer products in addressing the following overarching research topics: 1) NM characterization and quantification in environmental and biological matrices; 2) NM transformation in the environment and consequences for bioavailability and toxicity; 3) alternative methods to assess exposure; 4) influence of exposure scenarios on bioavailability and toxicity; 5) development of more environmentally realistic bioassays; and 6) uptake, internal distribution, and depuration of NMs. Research addressing these key topics will reduce uncertainty in ecological risk assessment and support the sustainable development of nanotechnology.

Toxicity Testing of Pristine and Aged Silver Nanoparticles in Real Wastewaters Using Bioluminescent Pseudomonas putida.

Impact of aging on nanoparticle toxicity in real matrices is scarcely investigated due to a lack of suitable methodologies. Herein, the toxicity of pristine and aged silver nanoparticles (Ag NPs) to a bioluminescent Pseudomonas putida bioreporter was measured in spiked crude and final wastewater samples (CWs and FWs, respectively) collected from four wastewater treatment plants (WWTPs). Results showed lower toxicity of pristine Ag NPs in CWs than in FWs. The effect of the matrix on the eventual Ag NP toxicity was related to multiple physico-chemical parameters (biological oxygen demand (BOD), chemical oxygen demand (COD), total suspended solids (TSS) pH, ammonia, sulfide and chloride) based on a multivariate analysis. However, no collection site effect was concluded. Aged Ag NPs (up to eight weeks) were found less toxic than pristine Ag NPs in CWs; evident increased aggregation and decreased dissolution were associated with aging. However, Ag NPs exhibited consistent toxicity in FWs despite aging; comparable results were obtained in artificial wastewater (AW) simulating effluent. The study demonstrates the potency of performing nanoparticle acute toxicity testing in real and complex matrices such as wastewaters using relevant bacterial bioreporters.

The MARINA Risk Assessment Strategy: A Flexible Strategy for Efficient Information Collection and Risk Assessment of Nanomaterials.

An engineered nanomaterial (ENM) may actually consist of a population of primary particles, aggregates and agglomerates of various sizes. Furthermore, their physico-chemical characteristics may change during the various life-cycle stages. It will probably not be feasible to test all varieties of all ENMs for possible health and environmental risks. There is therefore a need to further develop the approaches for risk assessment of ENMs. Within the EU FP7 project Managing Risks of Nanoparticles (MARINA) a two-phase risk assessment strategy has been developed. In Phase 1 (Problem framing) a base set of information is considered, relevant exposure scenarios (RESs) are identified and the scope for Phase 2 (Risk assessment) is established. The relevance of an RES is indicated by information on exposure, fate/kinetics and/or hazard; these three domains are included as separate pillars that contain specific tools. Phase 2 consists of an iterative process of risk characterization, identification of data needs and integrated collection and evaluation of data on the three domains, until sufficient information is obtained to conclude on possible risks in a RES. Only data are generated that are considered to be needed for the purpose of risk assessment. A fourth pillar, risk characterization, is defined and it contains risk assessment tools. This strategy describes a flexible and efficient approach for data collection and risk assessment which is essential to ensure safety of ENMs. Further developments are needed to provide guidance and make the MARINA Risk Assessment Strategy operational. Case studies will be needed to refine the strategy.